Caulobacter crescentus, a gram negative, aquatic bacterium adheres to surfaces via an adhesive extracellular polysaccharide holdfast. The long-term goal of this project is to study the mechanisms of holdfast synthesis and export regulation and how it is coordinated with the cell cycle as a model to understand ho w bacteria synthesize extracellular polysaccharides and attach to surfaces. This proposal focuses on HfsB, a homologue to the C-terminus of Wzc, the E. coli capsule export protein. I wish to elucidate how HfsB might be regulating the appearance of holdfast at a particular time in the cell cycle, and what other factors might be acting on HfsB to bring about this regulation. E. coli Wzc and its homologues are thought to regulate capsular polysaccharide (CPS) through their interaction with a multi-enzyme complex composed of components that catalyze polysaccharide synthesis and export and through their role as a tyrosine auto-kinase. When CPS export proteins such as Wzc are deleted or their ability to phosphorylate is eliminated by mutation, the synthesis of high molecular weight CPS is diminished as well. The specific aims include: (1) Determining the role of HfsB in holdfast production. HfsB does not have the typical consensus sequences of a tyrosine autokinase. Analysis of the effect of mutation in HfsB's putative ATP-binding site and anti-phosphotyrosine Western blots will determine if HfsB is a tyrosine autokinase. HfsB's interactions with other proteins involved in polysaccharide synthesis and export will be explored with tandem affinity purification (TAP). (2) Determining the timing of HfsA, HfsB, and HfsD localization. All three of these holdfast export proteins are present at similar levels throughout the entire cell cycle, suggesting localization at the appropriate cell pole is responsible for their activation. I will investigate if these proteins localize to the correct cell pole in time for holdfast production using both immunofluorescence and direct fluorescence microscopy. (3) It is likely that another protein acts on HfsB to bring about its regulation of holdfast synthesis and export. For instance, there are three developmental regulators whose mutation causes a holdfast synthesis deficiency. A multi-copy suppression screen and a traditional suppressor screen will be conducted to identify other proteins that might be involved in holdfast synthesis and export. Extracellular polysaccharides have an important role in disease. They increase resistance to antibiotics and to dessication of bacteria in medical biofilms, help bacteria evade the host immune system, and can be virulence factors themselves. Understanding how bacteria produce these polysaccharides is an important step in combating bacterial infection. [unreadable] [unreadable] [unreadable]